Trp-notrp-notrp-notrp-notrp-notrp-notrp-no: Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, , was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb3+-complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb3+ emission, I565/I540, was found to be 28.8-fold increased, and the dose-dependent enhancement of the I565/I540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a &sim;10-fold contrast window for the detection of NO. These unique properties allowed to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of was demonstrated by the visualization of NO in Daphnia magna. All of the results demonstrated that could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.

fig5: Effects of some ROS/RNS (1.0 mM) on the I565/I540 ratio (A) and the average luminescence lifetime (B) of TRP-NO (15 μM) in 0.05 M PBS buffer of pH 7.4 for 50 min.

Mentions:
The effects of some reactive oxygen/nitrogen species (ROS/RNS) on the I565/I540 ratio and luminescence lifetime of TRP-NO, were examined in 0.05 M PBS buffer of pH 7.4. As shown in Fig. 5, both the I565/I540 ratio and luminescence lifetime of TRP-NO did not show observable responses to the additions of different ROS/RNS including ClO–, H2O2, 1O2, ONOO–, ˙OH and O2–, while they were remarkably changed after TRP-NO was reacted with NO. These results indicate that the luminescence response of TRP-NO to NO is highly specific without significant interference from other ROS/RNS both under ratiometric time-gated and lifetime detection modes. In addition, the interference of some metal ions on the I565/I540 ratio of TRP-NO was further investigated in 0.05 M PBS buffer of pH 7.4 (Fig. S6†). The results revealed that the influence of common metal ions on the I565/I540 ratio of TRP-NO was negligible.

fig5: Effects of some ROS/RNS (1.0 mM) on the I565/I540 ratio (A) and the average luminescence lifetime (B) of TRP-NO (15 μM) in 0.05 M PBS buffer of pH 7.4 for 50 min.

Mentions:
The effects of some reactive oxygen/nitrogen species (ROS/RNS) on the I565/I540 ratio and luminescence lifetime of TRP-NO, were examined in 0.05 M PBS buffer of pH 7.4. As shown in Fig. 5, both the I565/I540 ratio and luminescence lifetime of TRP-NO did not show observable responses to the additions of different ROS/RNS including ClO–, H2O2, 1O2, ONOO–, ˙OH and O2–, while they were remarkably changed after TRP-NO was reacted with NO. These results indicate that the luminescence response of TRP-NO to NO is highly specific without significant interference from other ROS/RNS both under ratiometric time-gated and lifetime detection modes. In addition, the interference of some metal ions on the I565/I540 ratio of TRP-NO was further investigated in 0.05 M PBS buffer of pH 7.4 (Fig. S6†). The results revealed that the influence of common metal ions on the I565/I540 ratio of TRP-NO was negligible.

Trp-notrp-notrp-notrp-notrp-notrp-notrp-no: Rapid, multiplexed, sensitive and specific identification and quantitative detection of nitric oxide (NO) are in great demand in biomedical science. Herein, a novel multifunctional probe based on the intramolecular LRET (luminescence resonance energy transfer) strategy, , was designed for the highly sensitive and selective ratiometric and luminescence lifetime detection of lysosomal NO. Before reaction with NO, the emission of the rhodamine moiety in is switched off, which prevents the LRET process, so that the probe emits only the long-lived Tb3+ luminescence. However, upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from the Tb3+-complex moiety to rhodamine moiety occurs, which results in a remarkable increase of the rhodamine emission and decrease of the Tb3+ emission. After the reaction, the intensity ratio of the rhodamine emission to the Tb3+ emission, I565/I540, was found to be 28.8-fold increased, and the dose-dependent enhancement of the I565/I540 value showed a good linearity upon the increase of NO concentration. In addition, a dose-dependent luminescence lifetime decrease was distinctly observed between the average luminescence lifetime of the probe and NO concentration, which provides a &sim;10-fold contrast window for the detection of NO. These unique properties allowed to be conveniently used as a time-gated luminescence probe for the quantitative detection of NO using both luminescence intensity ratio and luminescence lifetime as signals. The applicability of for ratiometric time-gated luminescence imaging of NO in living cells was investigated. Meanwhile, dye co-localization studies confirmed a quite precise distribution of in lysosomes by confocal microscopy imaging. Furthermore, the practical applicability of was demonstrated by the visualization of NO in Daphnia magna. All of the results demonstrated that could serve as a useful tool for exploiting and elucidating the function of NO at sub-cellular levels with high specificity, accuracy and contrast.